Zinc Solid-State NMR Spectroscopy of Human Carbonic Anhydrase:  Implications for the Enzymatic Mechanism

Abstract

The pH dependence of the (67)Zn solid-state nuclear magnetic resonance spectroscopy of human carbonic anhydrase (CAII) has been investigated to characterize the nature of the fourth ligand. CAII, through the Zn(2+)-bound hydroxide, catalyzes the deceptively simple reaction: CO(2) + H(2)O <==> HCO(3)(-) + H(+). The accepted mechanism for CAII would predict that water would be bound to the Zn(2+) at pH 5 and hydroxide would be bound at pH 8.5. The measured values for the electric field gradient (EFG) or quadrupole coupling constant (Cq) for CAII are independent of pH within the limits of the experimental error, i.e., 9.8 +/- 0.2 MHz. The EFG interaction has been predicted by ab initio electronic structure calculations for water and hydroxide bound to the zinc, including various levels of hydrogen bonding. After comparing the predicted Cq's with the experimental values, we conclude that the species present from pH 5-8.5 is the hydroxide form. The NMR data presented here is not consistent with the accepted mechanism for CAII. We show that the NMR data is consistent with an alternative mechanism of CAII.

Towards Understanding Drugs on the Molecular Level to Design Drugs of Desired Profiles

“…As far as large structures are concerned, a striking example of NQR application was a direct observation of Zn 2+ in Carbonic Anhydrase (CA), the first enzyme whose activity was recognised to be related to the presence of Zn 2+ , which was postulated as the activator of bound H 2 O. CA, which commonly occurs in all mammalian tissues, plants, algae, and bacteria, is perhaps one of the best recognised metalloprotein. The 67 Zn QCC values, being sensitive to changes in the structure and bonding associated with water or hydroxide have been found independent of pH over the range of 5 to 8.5 [93], whereas, according to the hitherto commonly assumed model, Zn 2+ should be coordinated by H 2 O, and as a result, at these pH values the 67 Zn NQR spectrum should be three to five times broader than that at pH 8.5. This observation was in contradiction to the widely accepted mechanism and led to acceptance of an alternative one proposed by Merz, Hoffmann, and Dewar [94].…”

Crystal Chemistry of Zinc Quinaldinate Complexes with Pyridine-Based Ligands

“…Since then, over 1000 zinc metalloenzymes, covering all classes of enzymes, have been discovered [2][3][4][5][6]. The prevalent use of zinc in biological systems is due to its unique properties [3,5,[7][8][9][10][11][12][13][14]. Zinc(II) ion is characterized by a filled d subshell.…”

On the practical aspects of recording wideline QCPMG NMR spectra

“…Since all the intensity is allocated into sharp spikelets, a large gain in signal-to-noise ratio (S/N) is obtained at the expense of resolution, which is dictated by the spikelet separation. The (Q)CPMG pulse sequence has been applied on numerous occasions to facilitate the acquisition of dilute or unreceptive quadrupolar nuclei including 2 H [17], 14 N [18,19], 17 O [20,21], 25 Mg [9,[22][23][24][25], 27 Al [26][27][28], 33 S [29], 35 Cl [13,[30][31][32], 39 K [24,33,34], 47/49 Ti [35][36][37], 53 Cr [11], 55 Mn [38], 59 Co [39], 63/65 Cu [7], 67 Zn [8,24,[40][41][42][43][44][45][46][47], 87 Rb [48], …”